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Results: 4

1.
Figure 3

Figure 3. Systemic FasL neutralization enhanced lymphocyte activation and cytokine release into the airways after Af challenge. From: Systemic FasL Neutralization Increases Eosinophilic Inflammation in a Mouse Model of Asthma.

(A): Mice were sensitized and challenged as described and received Anti-murine FasL antibody (clone MFL4 or control hamster IgG). (B): Splenocytes were (harvested on Day 7 after Af challenge). 3H-thymidine uptake was compared between MFL4 and control antibody-treated mice 48 h later. (cpm): Count per minute in unstimulated (left panel) and in PHA stimulated (right panel) cells. (C): Dexamethasone dose response. n=4 spleens per group, *p<0.05, **p<0.01. (D): BAL was harvested on Day 1 after Af challenge. Mean ± SEM of n=16 (E): Correlation of soluble Fas ligand levels with IL-5, 9 and GM-CSF cytokines in the BAL harvested on Day 1 after Af challenge of MFL-treated mice.

Satish K. Sharma, et al. Allergy. ;67(3):328-335.
2.
Figure 4

Figure 4. FasL blockade with MFL4 enhanced eosinophilic airway inflammation. From: Systemic FasL Neutralization Increases Eosinophilic Inflammation in a Mouse Model of Asthma.

(A): Mice were sensitized and challenged with Af and treated with anti-murine FasL antibody as described. Total BAL cell counts were determined by Culter counter. (B): Absolute differential cell counts were assessed using the total cell counts and Giemsa-stained cytospin preparations (day 7 after challenge) (MP: macrophages; NP: neutrophils; EP: eosinophils; LC: lymphocytes) (C): Soluble FasL measured in the cell free BAL supernatant harvested on Day 7 after Af challenge (ELISA). (D): Absolute eosinophil count from BAL harvested on Day 7 after Af challenge. (A–D): Mean±SEM pooled from 3 separate experiments with n=14–22 mice per group. *p< 0.05, **p <0.01. (E/a.): Tissue eosinophils were labeled by an anti-major basic protein (MBP) monoclonal antibody. (E/b.): Eosinophil numbers were quantititated using a digital morphometric image analysis. (F): The number of submucosal eosinophils was determined by morphometric analysis and expressed as cell number per mm of epithelial basement membrane (BM). Mean±SEM, n=3 mice per group.

Satish K. Sharma, et al. Allergy. ;67(3):328-335.
3.
Figure 2

Figure 2. Eosinophil cell numbers in the airways negatively correlated with soluble FasL release. From: Systemic FasL Neutralization Increases Eosinophilic Inflammation in a Mouse Model of Asthma.

(A): FasL mRNA expression in the BAL cellular compartment (qPCR, normalized to GAPDH). Cells were harvested from mice sensitized and challenged with Af as described. Mean ± SEM; n=5 p<0.001 (two-way ANOVA). (B): Live BAL cells were stained in suspension with anti-FasL MFL3 or MFL4 clones on gelatinized chamber slides. Samples were obtained on Day 1 after Af challenge. (C): Formalin fixed, permeabilized tissue was stained for FasL [anti-murine polyclonal anti-FasL (N20)] or irrelevant rabbit IgG. (C/a–d.): Representative sections are shown for lungs harvested before (Day 0) and 1, 7 and 10 days after Af challenge of sensitized mice. (C/e.): Epithelial expression of FasL shown for the day 7 tissues. (C/f.): A corresponding frozen section labeled with the anti-FasL MFL4 clone. (C/g.): Ab control: irrelevant rabbit Ig. (D): Time course of soluble FasL protein expression in the BAL fluid and serum was determined by ELISA. Mean ± SEM; n=5 p<0.001 (two-way ANOVA). (E): Soluble FasL vs. BAL eosinophils correlation.

Satish K. Sharma, et al. Allergy. ;67(3):328-335.
4.
Figure 1

Figure 1. The peak of apoptotic cell numbers coincided with eosinophil influx into the airways but MBP+ eosinophils lacked TUNEL+ nuclei 1 day after a allergen challenge of sensitized mice. From: Systemic FasL Neutralization Increases Eosinophilic Inflammation in a Mouse Model of Asthma.

(A): BALB/c mice were intraperitoneally (i.p.) sensitized with Aspergillus fumigatus (Af) and sacrificed at the time points indicated by the black circles. (B): Absolute cell counts were assessed using Giemsa-stained cytospin preparations on Day 1 after Af challenge (MP: macrophages; NP: neutrophils; EP: eosinophils; LC: lymphocytes). (C): BAL absolute eosinophil count was assessed on Day 0 [prechallenge baseline], and 1, 7 and 10 days post Af challenge. (D): Th2 cytokines in the BAL were analyzed by a multiplex assay (Luminex, top panels). Eosinophil numbers were correlated with IL-4, 5, 9 and GM-CSF levels on Day 1 after Af challenge (Bottom panels; individual data points are shown). (E): Apoptotic cells in the airway were assessed by TUNEL (green) and anti-MBP antibody (red) labeling. (E/a.): Positive (DNAse treated) TUNEL control. (E/b.): Negative (no terminal transferase enzyme) control. (E/c.): Representative airway on Day 0 with no apoptotic cells. (E/d.): Representative airway on Day 1 with MBP positive (red) eosinophils and apoptotic cells having TUNEL positive (green) nuclei. (F): Submucosal TUNEL+ cells were quantititated using a digital morphometric technique. Results are expressed as Number of cells/mm basement membrane. (G): An MBP+ eosinophilic granulocyte is indicated by the red arrow. No MBP+ cells showed co-localization with TUNEL+ nuclei (green arrows); Aponecrosis (white arrow); (immersion oil, ×1000). (B–D & F): Mean±SEM; n=5 mice/time point.

Satish K. Sharma, et al. Allergy. ;67(3):328-335.

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